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  • 薛向阳1,张青锋1,黄玉富2,潘卫庆1,2*.K562细胞红系分化过程中miR-451表达的变化[J].第二军医大学学报,2008,29(5):0469-0473    [点击复制]
  • XUE Xiang-yang1,ZHANG Qing-feng1,HUANG Yu-fu2,PAN Wei-qing1,2*.Changes of miR-451 expression during erythroid differentiation of K562 cells[J].Acad J Sec Mil Med Univ,2008,29(5):0469-0473   [点击复制]
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K562细胞红系分化过程中miR-451表达的变化
薛向阳1,张青锋1,黄玉富2,潘卫庆1,2*
0
(1.同济大学传染病与疫苗研究所,同济大学医学院,上海 200092;2.第二军医大学基础部病原生物学教研室,上海 200433)
摘要:
目的:观察K562细胞红系分化过程中miR-451表达的变化,探讨miR-451在红细胞(RBC)形成过程中的作用。方法:应用Northern印迹法分析小鼠不同组织细胞(肝、骨髓、红细胞、白细胞)及人RBC、鸡RBC中miR-451的表达情况。采用氯高铁血红素(hemin,50 μmol/L)诱导K562细胞红系分化,诱导36 h后应用联苯胺染色及血红蛋白mRNA RT-PCR鉴定K562细胞红系分化结果,采用Northern印迹法及茎环RT-PCR分析K562细胞经hemin诱导不同时间点(24、48、72、96 h)miR-451表达的变化。结果:除红细胞外,小鼠白细胞、肝脏及骨髓Northern印迹均未检测到miR-451表达;人RBC及具有细胞核的鸡RBC亦检测到miR-451表达。小鼠及人RBC检测到2种miR-451,发现了1条较Sanger中心报道的序列3′端多1个尿嘧啶的miR-451序列。50 μmol/L hemin诱导后K562细胞出现红系分化,与诱导前相比,诱导36 h后联苯胺染色阳性细胞增多(P<0.05),γ、δ及ε血红蛋白表达增加(P<0.05)。Northern印迹未能检测到K562细胞诱导前后miR-451表达变化,但更敏感的茎环RT-PCR分析显示,诱导前K562细胞本身低丰度表达miR-451,hemin诱导24 h后,miR-451表达丰度开始增加,随着血红蛋白(δ-globin)表达增加,miR-451表达进一步增加,72 h达高峰。结论:miR-451是红系分化终末特异高表达的miRNA,在人及小鼠RBC中均存在相差1个碱基的2种序列,其在K562细胞红系分化过程中表达升高。
关键词:  K562细胞  miR-451  红细胞  细胞分化
DOI:10.3724/SP.J.1008.2008.00469
投稿时间:2007-12-14修订日期:2008-03-05
基金项目:
Changes of miR-451 expression during erythroid differentiation of K562 cells
XUE Xiang-yang1,ZHANG Qing-feng1,HUANG Yu-fu2,PAN Wei-qing1,2*
(1.Institute for Infectious Diseases and Vaccine Development,Medical College,Tongji University,Shanghai 200092,China;2.Department of Microbiology and Parasitology,College of Basic Medical Sciences,Second Military Medical University,Shanghai 200433)
Abstract:
Objective:To observe the expression of miR-451 during erythroid differentiation of K562 cells and investigate the role of miR-451 in erythroid differentiation.Methods: MiR-451 expression was analyzed in different tissues (the liver,bone marrow,erythrocytes,and white blood cells) of mice,human erythrocytes,and chicken red blood cells by Northern blotting.Erythroid differentiation of K562 cells was assessed by DAB staining and RT-PCR of heamoglobin mRNA before and 36 h after hemin induction (50 μmol/L).The expression of miR-451 in K562 cells was further explored by Northern blotting and stem-loop RT-PCR before and 24,48,72,and 96 h after hemin induction (50 μmol/L).Results: Expression of miR-451 was only identified in the erythrocytes,not in white blood cells,hepatic cells or bone marrow of mice.MiR-451 expression was also detected in human erythrocytes and chicken erythrocytes with nuclei.Two bands were detected in the human and mouse erythrocytes by Northern blotting,indicating that,in addition to the one reported by Sanger’s miRBase,there was another miR-451 sequence which had additional uracil residue in 3′ terminal.Hemin induced differentiation of K562 cells and DAB staining showed more positive cells after induction (P<0.05); the expression of γ, δ and ε-globin mRNA was also increased after treatment with hemin(P<0.05).Although Northern blotting revealed no changes in miR-451 expression in K562 cells after hemin induction,more sensitive stem-loop RT-PCR showed that miR-451 expression, which maintained at lower level in un-induced K562 cells, was increased during erythroid differentiation 24 h after hemin induction.With the upregulation of δ-globin protein,the expression of miR-451 reached its peak 72 h later.Conclusion: miR-451 is specifically and highly expressed in erythroid terminal differentiation.Two different sequences of miR-415 (one with and additional uracil residue) are present in the human and mouse erythrocytes,and their expression is elevated during the erythroid differentiation of K562 cells.
Key words:  K562 cells  miR-451  erythrocytes  cell differentiation